Intracellular protein degradation is a highly regulated process in which proteins are first targeted for degradation by conjugation to ubiquitin, a 76 amino acid polypeptide. Ubiquitinated proteins are then recognized by the 19S regulatory domain of the 26S proteasome. Through a series of ATP hydrolysis-dependent processes, targeted proteins are deubiquitinated and threaded into the core proteolytic complex, the 20S proteasome, where they are degraded into small peptides. Interestingly, exposure of cells to stimuli, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS), induces the synthesis of certain catalytic subunits (LMP2, MECL-1 and LMP7) that together are incorporated into alternative proteasome form, known as the immunoproteasome.
The immunoproteasome, as compared to the constitutive proteasome, has an enhanced capacity to generate peptides bearing hydrophobic and basic amino acids at their C-termini, and a reduced capacity to produce peptides bearing acidic residues at their C-terminus. Consequently, the spectrum of the produced peptides is shifted towards peptides which associate with MHC class I molecules with increased affinity, implicating a major role in antigen presentation. Immunoproteasome may be involved in some pathological processes, such as diabetes and autoimmune diseases. Therefore, development of immunoproteasome-specific inhibitors would be useful to investigate the role of immunoproteasome and to determine whether immunoproteasome is a potential target for development of pharmaceutical agents.

Anti-tumor natural products epoxomicin (1) and eponemycin (2) are members of linear peptides containing α′,β′-epoxyketone pharmacophore and have been shown to exert their anticancer activity through proteasome inhibition. Of particular interest was the finding that, despite structural similarities, epoxomicin (1) and dihydroeponemycin (3), an active derivative of eponemycin, differ in their proteasome subunit binding specificity.
Moreover, unlike other classes of proteasome inhibitors that show non-target specificity, the epoxyketone proteasome inhibitor is shown to be highly specific for the 20S proteasome. The crystal structure of the yeast 20S proteasome complexed with epoxomicin revealed that the unique specificity of epoxyketone pharmacophore is contributed to the formation of an unusual 6-membered morpholino ring between the amino terminal catalytic Thr-1 of the 20S proteasome and the (α′,β′-epoxyketone pharmacophore of epoxomicin, as shown in the mechanism below.

In addition, it has been shown that dihydroeponemycin (3) targets the subunits of both constitutive proteasome and immunoproteasome, whereas epoxomicin (1) preferentially labels the catalytic subunits of the constitutive proteasome. Recent studies indicated that the ability of dihydroeponemycin to bind immunoproteasome subunits is attributed to the P3 isooctanoic moiety of dihydroeponemycin but not the hydroxyl groups in the P2 and P1′ positions (see scheme 1). Therefore, isooctanoic-based dihydroeponemycin analogue (4) or other dihydroeponemycin analogues having a linear hydrocarbon group at the P3 position may provide an opportunity for the development of immunoproteasome-specific inhibitors. However, a simple and practical approach for the synthesis of dihydroeponemycin has yet to be developed. Particularly, the lack of the efficient synthetic approach for the hydroxymethyl-substituted enone motif has been a major obstacle for efficient synthesis of dihydroeponemycin and their P1′ derivatives (4).
Over the years, a number of elegant synthetic strategies for the synthesis of eponemycin and dihydroeponemycin have been developed. A key step in the synthesis is the preparation of hydroxymethyl-substituted enone 10 (Scheme 2). In several earlier approaches, the enone 10 was prepared from the reaction of dilithio reagent 8 with the corresponding aldehyde (Scheme 2). However, low yields and extra steps involving protection, oxidation and deprotection of OH groups prevented large scale preparation. In similar approaches, the Weinreb-type amide derivatives treated with dilithio reagent 8 did not yield the desired hydroxymethyl-substituted enone 10. More recently, new synthetic approaches have been developed based on the cinchona alkaloid-catalyzed Baylis-Hillman type reactions that yield the intermediate 7 or Stille coupling of Fmoc-Leu-Cl with n-tributylvinyltin followed by modified Baylis-Hillman reaction (Scheme 2).

However, multiple steps and low yields associated with these approaches may not be ideal for efficient derivatization or construction of small library of dihydroeponemycin analogues for screening immunoproteasome-specific inhibitors.
Over the past decades, the proteasome has emerged as a major player in many important signaling processes such as cell cycle progression, inflammatory responses and development. In particular, the fact that the orderly destruction of cell cycle regulatory proteins is critical to the control of cellular processes associated with cancer has led to development of proteasome inhibitors as anti-cancer agents, leading to a recent FDA approval of the first proteasome inhibitor bortezomib for multiple myeloma. Typically, more than 80% of cellular proteins are targeted for recognition and subsequent degradation by the proteasome via the attachment of multiple ubiquitin molecules.
The 20S core has a four-stacked ring structure with seven different subunits in each ring. The two inner rings each contain three catalytically active β subunits. The non-catalytic two outer a rings form a gated channel for unfolded protein entry and a base for the regulatory complexes (19S or 11S), which provide the specificity of the polypeptide recognition.
The 20S proteasome has been shown to exhibit three major activities: a chymotrypsin-like (CT-L) activity that cleaves after large hydrophobic residues, a trypsin-like (T-L) activity that hydrolyzes after basic amino acids, and a caspase-like (C-L) activity that cleaves after acidic amino acids. Two other less-characterized catalytic activities have also been ascribed to the proteasome: BrAAP, which cleaves after branched-chain amino acids, and SNAAP, which cleaves after small neutral amino acids. Thus far, while most efforts are directed to develop proteasome inhibitors against chymotrypsin-like activity, a few studies have been successful to design inhibitors targeting other proteasomal activities, such as caspase-like and trypsin-like activities. Although the CT-L activity of the proteasome has been shown to be largely responsible for the proteolytic function of the proteasome in vivo and in vitro, the contribution of other major activities remains to be determined.
While the immunoproteasome is widely known to play a major role in MHC class-I antigen presentation, it is believed not to be solely responsible for antigen presentation as the constitutive proteasome also generates immunogenic epitopes.
Recently, intense investigation on the role of immunoproteasome in cells from non-immune system has been initiated based on a number of studies indicating that immunoproteasome subunits may be implicated in some pathological processes, such as hematological cancers, autoimmune diseases and neurodegenerative diseases. For example, a high level of immunoproteasome has been detected in neurodegenerative human brains, whereas the human brain has been historically considered as an immunologically privileged organ. Specifically, it has been shown that the immunoproteasome is more highly expressed in the brains of Alzheimer's disease (AD) than in brains of non-demented elderly, whereas its expression in young brains is negligible or absent. In addition, some studies indicated that the immunoproteasome may be involved in Huntington's disease (HD) neurodegeneration. Multiple myeloma is also known to express a high level of immunoproteasome due to its bone marrow microenvironment where it replicates. Recently bortezomib (VELCADE®), the first proteasome inhibitor was approved by the FDA for the treatment of multiple myeloma. Despite this remarkable advancement, its clinical use is severely limited due to drug-related toxicities. Given this, specific inhibition of immunoproteasome should allow selective killings of multiple myeloma cells while sparing other cells in body that are lacking or minimally expressing the immunoproteasome.
Despite the potential role of immunoproteasome in these pathological disorders, its functions are still not clearly understood. Currently, there are no immunoproteasome specific inhibitors which are therapeutic agents, targeting the immunoproteasome. Furthermore, the exact role of immunoproteasome in pathogenesis is not clearly understood, due largely to the lack of an appropriate molecular probe.
Although some proteasome inhibitors currently exist that selectively target the immunoproteasome, and a sequence comparison of catalytic subunits from the constitutive and immunoproteasomes exhibits a high homology, structural information about active sites of immunoproteasome are not known to date, hindering prior efforts towards the design of immunoproteasome-specific inhibitors via rational design approach, to be therapeutic agents.

Two natural product proteasome inhibitors, epoxomicin and eponemycin, are members of the α′,β′-epoxyketone linear peptide family. It has been previously shown that, despite structural similarities, epoxomicin (1) and dihydroeponemycin (2), an active derivative of eponemycin, considerably differ in their proteasome subunit binding specificity. For example, dihydroeponemycin preferentially labels the catalytic threonine residues of immunoproteasome subunit LMP2 and to a lesser degree, the constitutive proteasome subunit X and immunoproteasome subunit LMP7. On the other hand, epoxomicin covalently modifies the N-terminus catalytic threonine residues of both the constitutive proteasome (X & Z) and immunoproteasome (LMP7 & MECL1) to a similar extent. It has been shown that a relatively higher specificity of dihydroeponemycin towards the immunoproteasome subunits as compared to epoxomicin is due to a linear hydrocarbon residue at the N-terminus (i.e., isooctanoic group). Recently, it has been shown that serine at the P2 site can be replaced with alanine while maintaining the same subunit binding pattern as dihydroeponemycin. More interestingly, careful analysis of other reports indicates that a residue at the P1′ site may be an important determinant for immunoproteasome subunit binding.